A healthy heart is vital for human life and well-being. Congenital heart disease affects ~1% of newborns and cardiovascular disease is the leading cause of death for adult men and women. Treatment of these diseases will rely on understanding their origin, and therefore on our detailed comprehension of normal heart development and heart function. There is currently a fundamental gap in knowledge about the mechanisms of how cardiovascular subtypes, such as atrial and ventricular myocytes, epicardium, endothelium, and smooth muscle cells are specified during development. This lack of detailed understanding of development and disease prevents the treatment of congenital heart disease, and hinders the translational approach of generating defined cell types from human pluripotent stem cells (hPSCs) for cell replacement therapies. Using genetic lineage-tracing approaches in the mouse, we have identified a novel progenitor population that is specified early during gastrulation and gives rise to ventricular cardiomyocytes and epicardium, but not atrial cells. We have demonstrated the transient existence of these cells during early development and our ability to isolate them from the embryo using a lineage-tracing model system. During the course of this project we will study the clonal lineage contribution potential o this ventricular-specific progenitor population during cardiac development using fate-mapping studies. Cell sorting and next generation gene expression analysis will be utilized to identify the molecular mechanisms that govern early atrial-ventricular specification. Furthermore we will investigate the functional importance of the newly identified ventricular-specific progenitor population during cardiac development using genetic loss of function strategies. Finally, we will translate these concepts to the hPSC system. The long-term goal of our studies is to understand the developmental principles of cardiac cell fate specification in vivo, and to apply these principles to the PSC model system in order to generate defined cardiovascular cell types in vitro. The overall objective of our application is to identify the mechanisms that direct atrial ventricular cell fate specification during early development and to translate them to efficiently generate human atrial and ventricular cardiomyocytes from PSCs. Such protocols do not currently exist. Our central hypothesis is that atrial-ventricular specification occurs early durin development and that the respective progenitor populations are characterized by specific gene expression. In conclusion, studying this previously unidentified progenitor population during heart development is important because it will advance our understanding of heart development and disease by uncovering a novel concept of early cardiac cell specification. Ultimately, such knowledge has a broad translational importance in the understanding of congenital heart disease as well as the development of therapeutically relevant and safe cell populations from hPSCs, with the ultimate goal to treat the growing number of patients with cardiovascular disease in the United States and beyond.